Cleaning a mortar/pestel

[Lawrence R Hale]

I am going to try to work with plant DNA for the first time. I will be
doing a mortar/pestel grind of leaf tissue under liquid nitrogen, as
specified by the protocol I am following.

My question concerns the mortar and pestel. What is the correct way to
clean the mortar and pestel between grinds so that I don't contaminate
one sample with tissue from another?

Thanks,

Larry Hale, Univ. of PEI
lh...@upei.ca

[R. John Lye]

Lawrence R Hale wrote:
> My question concerns the mortar and pestel. What is the correct way to
> clean the mortar and pestel between grinds so that I don't contaminate
> one sample with tissue from another?

We don't clean them between samples, we simply use a fresh one for each
sample. Of course, that does limit how many samples one can process at
a time.

--
John Lye
rj...@Virginia.edu

[Jim Kami]

Hi,
    How clean you need to be depends upon the final use of the DNA. For RFLP work, we normally grind 5-10 gms of leaves and just wipe the mortar out with a dry cloth (wet cloth will freeze to the mortar) between samples. Because we get 50-100 ug of DNA at the end of the procedure, any contaminating DNA will be so small in relation to the desired sample it's considered negligible. Also, since we often have to do several hundred samples at a time, it's the only practical method.
    For PCR based work however, we do use clean mortars for each sample, since even minuscule contamination might be amplified. (Over the years we have acquired about 50 mortars so this is still somewhat practical, albeit a bit slower).
    Two tips: 1) to clean the mortars we wait for them to warm up to room temp. and then scrub them out with a ScotchBrite pad and 0.1% SDS, rinse well with DI H2O and dry. Don't autoclave or use a commercial glasswasher. We have split an awful lot of mortars that way. The thick ceramic can't handle the temp changes for some reason.
2) If you pre-chill the mortar and pestle in a -80C freezer for an hour or two, you don't have to keep adding LN2 to keep the tissue cold. Makes a lot less mess when the ground tissue boils out with the LN2.

Jim Kami                              email: jak...@ucdavis.edu
Department of Agronomy                Tel: (530) 752-9982
Hunt Hall Rm 272                      Fax: (530) 752-4361
University of California
1 Shields Avenue
Davis, CA 95616-8515 USA

      "Why does Common Sense always seem to be the least common sense ?!?"
 

Lawrence R Hale wrote:

I am going to try to work with plant DNA for the first time. I will be
doing a mortar/pestel grind of leaf tissue under liquid nitrogen, as
specified by the protocol I am following.

My question concerns the mortar and pestel. What is the correct way to

clean the mortar and pestel between grinds so that I don't contaminate
one sample with tissue from another?

Thanks,

[Savita Shah]

How many gm or milligrams of tissue you are using? You can get 10 mortar
and pestle (small ones) and use them once .. and as you finish grinding
one sample put them in small tub of warm water (with commercial
detergents like Dawn/joy, palmolive.. ) anyways.. you will probably have
to incubate you samples in extraction buffers for 10 to 15 min.. and so
that way you get a break from grinding samples.. Wash the mortor and
pestles and rinse with 70% Ethanol and use them for teh next batch of
samples!!!

[Vellanoweth's Lab]

I often isolate plant RNA with a mortar and pestle and liquid nitro. I
use a different mortar and pestle for each sample and I only do about 10
samples at a time. It's not really practical to clean them out in between
samples. After I'm done, I scrub out the mortars with a scrub brush and
SDS soap solution. Then I dry with ethanol and bake for 2 hours at 180
(that's for RNAse destruction - not really necessary for DNA work
probably).

Janel Wheeler
Vellanoweth Biochemistry Lab
California State University, Los Angeles
jwh...@calstatela.edu

[Michael L. Sullivan]

>I often isolate plant RNA with a mortar and pestle and liquid nitro. I
>use a different mortar and pestle for each sample and I only do about 10
>samples at a time. It's not really practical to clean them out in between
>samples. After I'm done, I scrub out the mortars with a scrub brush and
>SDS soap solution. Then I dry with ethanol and bake for 2 hours at 180
>(that's for RNAse destruction - not really necessary for DNA work
>probably).
>

Not really necessary for RNA work either! I think you'd be better off not
baking. I think baking only serves to make the mortars break faster. If
you think about it, this step is not needed. By grinding your plant
material in the mortar and pestal, you will be releasing far more RNase
activity than you get rid of by baking the mortar! And that's why the
first wet step in most RNA preps is to put the tissue into a buffer with
GTC or some other strong denaturant.

Mike

Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
---